Compositions containing preen oil and methods of use thereof

ABSTRACT

Described herein are methods of use of purified preen oil, food and feed compositions containing purified preen oil, food and feed additives containing purified preen oil, and pharmaceutical compositions containing purified preen oil. Methods include treatment of chronic joint inflammation in humans and animals by orally administering purified preen oil. Also included are methods of feeding a growing fish by feeding the feeding the growing fish purified preen oil in amounts effective to improve survival and/or stimulate growth in the growing fish. Further included are methods of feeding live prey organisms purified preen oil, and feeding the live prey organisms to growing fish.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/931,289filed on Nov. 3, 2015, which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under 18-CRHF-0-6055awarded by the USDA/NIFA. The government has certain rights in theinvention.

FIELD OF THE DISCLOSURE

The present disclosure is related to compositions and methods for theoral administration of naturally occurring oils, particularly food andfeed compositions.

BACKGROUND

The exudate from the preen (or uropygial) gland found at the base of thetail of most birds, is applied to the plumage during preening. Preengland exudate has a lipid fraction and a non-lipid fraction containingproteins, inorganic salts and cell fragments. The lipid fraction (hereinreferred to as preen oil) is made up of monoester, diester and triesterwaxes (long-chain carbon molecules which contain one, two or three esterbonds). Preen oil isolated from turkey preen glands has been shown, forexample, to include the fatty acids C10:0, C12:0, C14:0, C16:0, C17:0,C18:0, C19:0 and C20:0. In vitro assays have suggested that C19:0 (19:0or nonadecanoic acid) has possible anti-inflammatory, anti-tumor, andproliferous cell inhibitory effects.

There is increasing interest in natural substances that have healthbenefits in medicine and animal agriculture. Preen glands are includedin segregated tissue from animal processing (STFAP). Currently, STFAP isdiscarded or rendered with other wastes during meat harvest andprocessing, and can be separated from a larger stream of waste andby-products. The oil from the animal tissue can be easily removedthrough mechanical means (dissection) and extracted using solvents. Itis thus desirable to isolate preen oil from preen glands that wouldotherwise be part of the waste stream of animal processing and toidentify uses for the purified preen oil.

What is needed are methods of use of natural substances such as preenoil to provide health benefits in humans and animals.

BRIEF SUMMARY

In one aspect, a method of treating an individual in need of treatmentfor chronic inflammation comprises orally administering to theindividual a composition comprising a therapeutically effective amountof purified preen oil.

In another aspect, a food or feed composition comprises 0.01 to 10 wt %of purified preen oil, and a basal food composition. Also includedherein is a food or feed additive composition comprising 0.1 to 99 wt %of purified preen oil.

In another aspect, a method of feeding a growing fish comprises feedingthe growing fish a fish food composition comprising an effective amountof purified preen oil to improve survival and/or stimulate growth in thegrowing fish.

In another aspect, a method of reducing a risk of hypoxia in a fish inaquaculture, comprises feeding the fish a fish food compositioncomprising an effective amount of purified preen oil to reduce the riskof hypoxia in the fish in aquaculture.

In yet another aspect, an aquaculture fish feed composition comprises0.01 to 10 wt % of purified preen oil, and a basal aquaculture fish feedcomposition. Also included herein is an aquaculture fish feed additivecomposition comprising 0.1 to 99 wt % purified preen oil.

In a still further aspect, a method of feeding live prey organisms foruse as food in aquaculture comprises feeding the live prey organisms adiet comprising an effective amount of purified preen oil to increase apreen oil lipid content of the live prey organisms.

In a yet further aspect, a pharmaceutical composition comprises 0.1 to99 wt % of purified preen oil, and a pharmaceutically acceptableexcipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fatty acid content of preen oil purified from the preenglands of layers and young broilers.

FIG. 2 shows the clinical arthritic score over a 63-day period for micesupplemented with 3% preen oil compared to 3% corn oil.

FIG. 3 shows the reduction in arthritic severity over a 63-day periodfor mice supplemented with 3% preen oil compared to 3% corn oil.

FIG. 4 shows the feed fatty acid composition for a 3% corn oil and a 3%preen oil mouse diet.

FIG. 5 shows mouse paw IL-1β in non-arthritic mice, arthritic micesupplemented with 3% canola oil, and arthritic mice supplemented with 3%preen oil.

FIG. 6 shows mouse paw IL-6 in non-arthritic mice, arthritic micesupplemented with 3% canola oil, and arthritic mice supplemented with 3%preen oil.

FIG. 7 shows the average mouse weight gain over a 63-day period in 8week old, weight matched mice fed either 3% canola oil diet or 3% preenoil diet and weighed weekly.

FIG. 8 shows the effects of 2% dietary preen oil on the growth of larvalfathead minnows.

FIG. 9 shows the effects of 2% dietary preen oil on the survival oflarval fathead minnows.

FIG. 10 shows the survival rates of juvenile walleyes subjected to acutehypoxia.

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

Described herein are methods of orally administering purified preen oilto provide health benefits in humans and animals. The health benefits oforally administered preen oil had not previously been reported. It hasbeen unexpectedly discovered herein that oral administration of purifiedpreen oil has anti-inflammatory and other health benefits in animals. Inan aspect, orally administered purified preen oil reducesdisease-associated chronic inflammation. In another aspect, orallyadministered purified preen oil reduces chronic joint inflammation suchas that associated with rheumatoid arthritis (RA). In yet anotheraspect, oral administration of purified preen oil to larval and growingfish, particularly fish growing in aquaculture, results in decreasedmortality, improved health, and increased growth.

As used herein, purified preen oil is defined as the lipids isolatedfrom the preen gland of fowl, such as chickens and turkeys. Purifiedpreen oil is substantially free of the non-lipid fraction of preenexudate containing proteins, inorganic salts and cell fragments.Substantially-free means that the preen oil contains less than about 61wt % of non-lipid components, specifically less than about 50 wt %non-lipid components, and more specifically less than about 20 wt %non-lipid components. While there is some variability in the compositionof preen oil depending upon the source and the purification method, ingeneral, preen oil contains 6:0, 7:0, 8:0, 9:0, 10:0, 11:0, 12:0, 13:0,14:0, 15:0, 16:0, 17:0, 18:0, 18:1 C9, 19:0, 18:2 and 20:0 fatty acids.

In one aspect, a method of treating an individual in need of treatmentfor chronic inflammation comprises orally administering to theindividual a composition comprising a therapeutically effective amountof purified preen oil. As used herein, chronic inflammation is not acuteor injury-related and is associated with dysregulation of thepro-inflammatory cytokines IL-1 and IL-6. Inhibition of IL-1 and IL-6 bythe administration of preen oil produces broad anti-inflammatoryeffects. In one aspect, the chronic inflammation is associated with adisease wherein inhibition of the pro-inflammatory cytokines IL-1 andIL-6 reduces disease-associated chronic inflammation. In another aspect,the chronic inflammation is chronic joint inflammation which isgenerally associated with pain lasting for weeks or even years, orchronic joint inflammation associated with the natural aging process. Ina still more specific aspect, the chronic inflammation is associatedwith a disease associated with chronic joint inflammation such asarthritis.

Individuals within the scope of the present methods include mammals andnon-mammals such as fish and birds. Mammals include humans, dogs,equines, and porcines and bovines, for example.

Human diseases wherein inhibition of the pro-inflammatory cytokines IL-1and IL-6 reduce disease-associated chronic inflammation includeautoinflammatory syndromes such as Cryopyrin-associated periodicsyndromes (CAPS), familial Mediterranean fever (FMF), Schnitzler'ssyndrome, adult onset Still's Disease (aoSD), antisynthetase syndrome,relapsing polychondritis, familial cold autoinflammatory syndrome (FACS,FCAS2), Muckle-Wells syndrome (MWS), dermatomyositis (DM), polymyositis(PM), sporadic inclusion body myositis (IBM), necrotizing autoimmunemyopathy (NAM), inflammatory bowel disease (IBD), post-infarctioninflammation, Tumor Necrosis Factor Receptor Associated PeriodicSyndrome (TRAPS), systemic autoinflammatory disease (SAID), NOMID/CINCA,PFAPA (periodic fevers with aphthous stomatitis, pharyngitis, andadenitis) syndrome, recurrent idiopathic pericarditis, Mollaretsyndrome, delayed pressure urticaria, pyoderma gangrenosum and acne(PAPA) syndrome, Deficiency of Il-1 Receptor Agonist (Il-1 RA) (DIRA),Majeed Syndrome, CARD14 mediated psoriasis (CAMPS), deficiency ofinterleukin 36 receptor antagonist (DITRA), Blau's syndrome, ChronicRecurrent Multifocal Osteomyelitis (CRMO), Synovitis, Acne, Pustulosis,Hyperostosis and Osteitis (SAPHO) Syndrome, HLA-B27 spondyloarthropathy,Sweet syndrome, generalized pustular psoriasis, Hallopeauacrodermatitis, Nakajo-Nishimura syndrome, Joint contractures, muscleatrophy, microcytic anemia and panniculitis-induced lipodystrophy (JMP)syndrome, Chronic atypical neutrophilic dermatosis with lipodystrophyand elevated temperature (CANDLE) syndrome, early-onset inflammatorybowel disease, neutrophilic panniculutis, Erythema nodosumandpanniculitis, Crohn's disease. Microphage activation syndromes (MAS),familial hematophagocytic lymphohistiocytosis, and Castleman's disease.

Chronic joint inflammation in humans is associated with rheumatoidarthritis (RA), systemic lupus erythematosus, reactive arthritis, lupusarthritis, arthritis associated with Sjogrens syndrome, systemic onsetjuvenile idiopathic arthritis (SOJIA), gout, pseudogout, osteoarthritis,psoriatic arthritis, peripheral joint inflammation associated withinflammatory bowel disease, Ankylosing spondylitis, reactive arthritis,age-related joint degeneration, or chronic Lyme disease, for example. Inone aspect, the individual is a human individual.

Orally administered therapeutically effective amounts of purified preenoil for the treatment of chronic inflammation in humans are 0.001 to 100g per day, specifically 0.025 to 25 g per day. In one aspect, preen oilis orally administered to a human in an amount of greater than or equalto 25 mg per day. Since preen oil is safe for consumption, there shouldbe no toxicity associated with the higher ranges. Higher ranges arepreferred for more rapid incorporation in tissues and low ranges forlong-term use.

In another aspect, the individual is a companion animal such as a dog,or a livestock animal such as cattle. Dogs, equines, porcines and cattlesuffer from diseases associated with chronic inflammation such asosteoarthritis, chondrodystropy, arthritis, osteochondrosis,spondylosis, intervertebral disk disease, diskopondylitis, degenerativearthropathy, hip dysplasia and chronic laminitis. Dogs are known tosuffer from chronic joint inflammation due to arthritis and pigs,horses, and cattle suffer from chronic joint inflammation due tolameness. Orally administered therapeutically effective amounts ofpurified preen oil for the treatment of chronic inflammation in dogs andcattle are 0.0001 to 450 g per day, specifically 0.001 to 145 g per day.These ranges are based on metabolic scaling where the low end would bethe minimal levels for a 1 Kg dog and the high range for a 750 Kgcattle. In a specific aspect the purified preen oil is orallyadministered in an amount of greater than or equal to 1 mg per day.

Exemplary compositions for the oral administration of purified preen oilinclude pharmaceutical compositions as well as food compositions such asnutraceutical food compositions, described in more detail below.

Low-grade chronic inflammation underpins many diseases affecting humanhealth such as RA and remains largely undiagnosed and undertreatedworldwide. Without being held to theory, it is believed that fatty acidssuch as those found in purified preen oil will accumulate in body cellsand tissues after oral consumption, thus purified preen oil is predictedto be an effective oral treatment for reducing damaging effects ofchronic inflammation, including chronic joint inflammation, such asjoint destruction in RA.

Specifically, the anti-inflammatory effects of dietary purified preenoil were studied in a model of chronic inflammation associated withdysregulation of the pro-inflammatory cytokines IL-1 and IL-6.Chronically inflamed arthritic mice over a 9 week period using thecollagen-induced arthritis model were used. As shown in the examples,the results from this pre-clinical animal study demonstrate the efficacyof purified preen oil as an oral anti-inflammatory agent. Similarresults are expected for the treatment of all types of chronicinflammation and diseases associated with chronic inflammation and morespecifically chronic joint inflammation such as arthritis.

Further included herein are feed/food compositions and feed/foodadditives comprising purified preen oil. As used herein, the term “foodcomposition” and “food additive” refer to compositions for ingestion bya human while “feed composition” and “feed additive” refer tocompositions for ingestion by an animal.

In an aspect, a food or feed composition comprises 0.001 to 50 wt %specifically, 0.01 wt % to 10 wt %, and more specifically 0.1 wt % to 5wt % of purified preen oil, wherein the food composition comprises abasal food composition and is suitable for ingestion by a human, and thefeed composition comprises a basal feed composition and is suitable foringestion by an animal. In one aspect, a basal food or feed compositionis a nutritionally complete food or feed composition.

As used herein, the terms “food” and “feed” broadly refer to a material,liquid or solid, that is used for nourishing a human or animal. Feedcompositions, for example are used for sustaining normal or acceleratedgrowth of an animal including newborns or young and developing animals.The terms include a compound, preparation, mixture, or compositionsuitable for oral intake by a human or animal. Feeds are suitable forherbivorous mammals such as cattle, horses, sheep and goats; for fishand poultry; or for companion animals, for example. A food/feedcomposition comprises a basal food/feed composition and one or morefood/feed additives. The term “basal food/feed composition” refers to afood/feed composition combinable with purified preen oil describedherein. The term “feed/food additive” as used herein refers tocomponents included in small quantities for the purpose of fortifyingbasic feed/food with nutrients, stimulants, medicine, or to promote feedintake or alter metabolism. As a general rule, feed/food additive areadded to a basal feed/food composition in an amount of 0.01 to 10 wt %based on the weight of the basal feed/food composition. Feed/foodadditives include pre-mixes of biological compositions, or in thepresent disclosure, compositions containing purified preen oil andoptionally at least one additional edible component.

Basal feed compositions may include components such as a protein source,a grain, flavor compositions, vitamins, minerals, preservatives, andcombinations thereof. Basal feed compositions can be suitable foringestion by a target animal.

An animal feed may further include optional ingredients includingvitamins, minerals, antibiotics, lipids, carbohydrates, proteins,antioxidants, and amino acids.

Exemplary vitamins include Vitamin A, Vitamin B, Vitamin D, Vitamin E,and Vitamin K. Exemplary minerals include calcium, phosphorus, sodium,potassium, magnesium, chlorine, cobalt, iodine, iron, manganese, copper,molybdenum, zinc and selenium. Common mineral supplements used inpoultry feed, for example, include limestone, bone meal, oyster shell,sodium chloride, dicalcium phosphate, manganese sulphate, potassiumiodide, and superphosphate.

In some embodiments, one or more antibiotics may be included in theanimal feed along with the feed additive. Exemplary antibiotics includepenicillin, streptomycin, tetracyclines, zinc bacitracin and aureomycin.

Exemplary lipids include oil seeds, oils and lipids derived from plantsor animals. Sources of oilseeds, oils and lipids include corn, soybean,cotton, lupin, peanut, sunflower, canola, sesame seed oil, olive oil,copra and coconut oil, palm kernels and palm oil, casein, butterfat,lard, fish oils, linseed and oil, tuna oil, tallow and yellow grease,and mixtures thereof.

Exemplary carbohydrates include starch, cellulose, pentosans, othercomplex carbohydrates, corn, milo, barley, rye, oats, wheat, wheatmiddlings, and various grain-by-products.

Exemplary sources of protein include protein obtained from meat meal orfish meal, liquid or powdered egg, fish solubles, whey, milk protein,rice, milo, millet, corn, oats, barley, wheat, rye, wheat bran and/ormiddlings, soybeans, sesame seeds, peas and beans, sunflower seeds,wheat germ, alfalfa seed, flaxseed, yeast, earthworms, and fish.

Exemplary amino acids include arginine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, threonine, tryptophan, valine,tyrosine ethyl HCl, alanine, aspartic acid, sodium glutamate, glycine,proline, serine, cystein ethyl HCl, and analogs, and salts thereof.

Exemplary antioxidants include beta-carotene, Vitamin E, Vitamin C, andtocopherol, or synthetic antioxidants.

In another aspect, included herein is a food composition suitable foradministration to humans containing a basal food composition andpurified preen oil. As used herein, the term “food” or “food product”refers to a food suitable for consumption by humans. The “food product”may be a prepared and packaged food (e.g., milk, yogurt, or cheese).Food products include prepared food products. “Prepared food product”means a pre-packaged food approved for human consumption.

An exemplary food product is a dietary supplement such as a powder,liquid concentrate, bar or shake. Powders and concentrates can be addedto a liquid such as water or milk for consumption. In addition topurified preen oil, the dietary supplement can comprise additionalcomponents such as vitamins, minerals, amino acids and protein sources.

In another aspect, a functional food or feed additive compositioncomprises 0.001 to 99 wt %, specifically, 0.01 wt % to 95 wt % ofpurified preen oil, wherein the functional food additive compositioncomprises a basal functional food additive composition and is suitablefor ingestion by a human, and the functional feed additive compositioncomprises a basal functional feed additive composition and is suitablefor ingestion by an animal. A 100% purified preen oil food or feedadditive composition could, for example, be in the form of a capsule. Asused herein, a functional food/feed additive is a functional ediblesubstance that is not consumed as a food itself, but that is added tofood to provide or maintain nutritive value. Essentially, a functionalfood/feed additive is a dietary supplement that is added directly to afood or feed composition to improve the properties of the food or feedcomposition or the health of the animal. Basal functional food/feedadditives include, vitamins, minerals enzymes, essential oils,acidifiers prebiotics, probiotics, botanical products yeast and yeastfractions, as well as carriers.

In another aspect, included herein are pharmaceutical compositionscomprising preen oil. For example, a pharmaceutical compositioncomprises 0.001 to 99 wt %, specifically 0.1 wt % to 99 wt % of purifiedpreen oil, and a pharmaceutically acceptable excipient.

Tablets and capsules for oral administration may be in unit dose form,and may contain conventional excipients such as binding agents, forexample syrup acacia, gelatin, sorbitol, tragacanth, orpolyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch,calcium phosphate, sorbitol or glycine; tabletting lubricant, forexample magnesium stearate, talc, polyethylene glycol or silica;disintegrants for example potato starch, or acceptable wetting agentssuch as sodium lauryl sulphate. The tablets may be coated according tomethods well known in normal pharmaceutical practice. Oral liquidpreparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavoring or coloring agents.

In another aspect, a method of feeding a growing fish comprises, feedingthe growing fish a fish food composition comprising an effective amountof purified preen oil to improve survival and/or stimulate growth in thegrowing fish. As used herein, the term fish includes finfish andshellfish such as shrimp. The term growing fish means fish that areincreasing in length and weight in time. Exemplary growing fish arelarval and juvenile fish. New methods to improve fish growth andsurvival are needed, and in particular additives for fish feed that canimprove survival and/or growth and that are stable to the fish feedproduction process. Growth can be measured as the length of the fish,such as the average length of fish in a population.

In a specific aspect, the fish is in aquaculture. As used herein,aquaculture means the active cultivation of aquatic organisms undercontrolled conditions. Aquaculture systems use water as the medium forcultivation. An aquaculture system must provide clean and oxygenatedwater to support the cultivated organisms as well as a means to removedeoxygenated water and wastes. As used herein, aquaculture includes bothmarine and freshwater aquaculture. Typical aquaculture systems includeholding tanks and means for filtering, dissolved gas control, andtemperature control. Aquaculture typically requires a preparedaquaculture feed composition to meet dietary requirements of thecultured animals.

Basal aquaculture fish feed compositions, for example, contain a proteinsource such as fish meal. Due to problems securing fish to produce fishmeal and the depletion of fish stock for feeding fish, attempts havebeen made to supplement fish foods with other proteins. Soy protein iscommonly used as a protein source in basal fish feeds, however soyprotein induces gut inflammation and increases proinflammatorycytokines. These cytokines are linked to decreased food intake andgrowth. Researchers have been trying to find ways to counteract theproinflammatory effects of certain alternative fish foods. An advantageof the use of purified preen oil in fish food compositions foraquaculture is that it reduces proinflammatory processes known to reducegrowth and can reduce the negative effects of commonly available proteinsources such as soy protein

High valued fish for aquaculture applications (carp, tilapia, hybridstriped bass, salmon, trout, catfish, yellow perch, walleye; marinespecies such as cod, cobia, sea bass, tuna, and sole; and shellfish suchas shrimp, scallops and oysters for example) may have poor larval growthand high rates of mortality, which is delaying the expansion of theaquaculture industry. In particular, there can be high mortality ratesof up to 95% at the larval stage. Inflammation of the gastrointestinaltract has been shown to slow growth in fish species. New methods toimprove fish growth are needed. Purified preen oil can be added to afish feed or a fish feed pre- or post-pellet formation and can either beadded on the diet or as a replacement for some of the lipid in the fishfood.

In experiments described herein, larval and fish food pellets served asa control or were coated with 2% preen oil. The fish were fed the twodiets in 4 tanks each and growth was measured by length increases. Theexperiment lasted 30 days. Larval fish fed the preen oil had a 27%increase in growth (P=0.02). In addition, preen oil improve survival infish exposed to hypoxic conditions, demonstrating that preen oil reducesthe susceptibility of the fish to stress and increases their chances ofsurvival in culture.

In another aspect, a diet containing purified preen oil was found toreduce hypoxia in fish in aquaculture that were subjected to hypoxicconditions. A method of reducing the risk of hypoxia in a fish inaquaculture comprises feeding the fish a fish food compositioncomprising an effective amount of purified preen oil to reduce the riskof hypoxia in the fish in aquaculture. In one aspect, the fish have beenexposed to hypoxic conditions or are at risk of being exposed to hypoxicconditions. Hypoxia is a known problem in aquaculture, particularly pondaquaculture, where fish are often raised at high densities. During theday when the algae in the ponds are photosynthesizing, there istypically enough oxygen in the ponds. At night, however, the algae startto respire and oxygen levels can drop dramatically. Some or all fishoften die overnight due to hypoxia, which is why many commercialaquaculture facilities rely on aerators to oxygenate the pondsovernight. The ability of preen oil to protect fish from hypoxia inaquaculture is a significant discovery.

In one aspect, the fish food composition comprises 0.01 wt % to 10 wt %,specifically, 0.1 wt % to 2 wt % of purified preen oil on a w/w basisand a basal fish feed composition. In a more specific aspect, includedherein is an aquaculture fish feed composition containing purified preenoil and a basal aquaculture fish feed composition. The aquaculture fishfeed composition comprises 0.01 wt % to 10 wt %, specifically, 0.1 wt %to 2 wt % of purified preen oil. Typical components in a basal fish feedor aquaculture fish feed composition include proteins and carbohydrates,based on fish meal, soybean meal or vegetable/corn meal, and may besupplemented with essential amino acids, proteins, peptides, vitamins,minerals, carbohydrates, fats, oils, and combinations thereof. Vitaminsinclude A, E, K, D₃, B₁, B₃, B₆, B₁₂, C, biotin, folic acid,panthothenic acid, nicotinic acid, choline chloride, inositiol, andpara-amino-benzoic acid. Minerals include salts of calcium, cobalt,copper, iron, magnesium, manganese, phosophorus, potasium, selenium andzinc. Other components may include, but are not limited to,antioxidants, beta-glucans, bile salt, cholesterol, enzymes, monosodiumglutamate, and the like. Basal aquaculture fish feed compositions areprepared products generally in the form of flakes, pellets or tabletsthat form a nutritionally complete diet for the aquacultured species.The purified preen oil can be added during production of the aquaculturefish feed composition so that it is substantially homogeneouslydistributed throughout the aquaculture fish feed composition, or thepurified preen oil can be added to a pre-prepared basal aquaculture fishfeed composition for example by top dressing.

In other aspect, soy is used as a protein source in a basal fish feedcomposition. Soybeans in the form soy flours, soy flakes, and soy mealgenerally have off-flavors that are unpalatable to aquacultured fish dueto their relatively higher antigenicity. Fish feed compositionscontaining purified preen oil may allow for the use of higher amounts ofsoy protein in fish food compositions.

The importance of providing a sufficient amount of lipids for successfulproduction of marine fish larvae is known in the art. The two main livefeeds used for marine fish larval production, rotifers and Anemia (brineshrimp nauplii), naturally lack certain long chain polyunsaturated fattyacids and highly unsaturated fatty acids must be supplemented to ensuresuccessful survival, growth and metamorphosis of the larvae. A majorsource of long chain polyunsaturated fatty acids for live feedsupplementation is fish oils. Saturated and monounsaturated fatty acidshave also been identified as major energy yielding nutrients in fish.U.S. Pat. No. 7,063,855 describes feeding lipids to aquacultural preyorganisms to improve the highly-unsaturated fatty acid content of thelive food.

Also included herein are aquaculture fish feed additive compositioncomprising purified preen oil, such as 0.1 to 99 wt % purified preenoil. The aquaculture fish feed additives optionally include an ediblecomponent in addition to the purified preen oil.

As an alternative to feeding fish a feed containing purified preen oil,fish can be fed live prey organisms that have been fed a diet containingpurified preen oil. In one aspect, a method of feeding live preyorganisms for use as food in aquaculture comprises feeding the live preyorganisms a diet comprising an effective amount of purified preen oil toincrease a preen oil lipid content of the live prey organisms. In anaspect, the diet for the live prey organisms comprises 0.1 wt % to 50 wt%, specifically 1 wt % to 10 wt % of purified preen oil based on thetotal weight of the diet. In another aspect, the preen oil fatty acidcontent of the live prey organisms that have been fed purified preen oilis 0.01 to 10% of total fatty acids of the prey.

In another aspect, a method of feeding a growing fish such as a larvalor juvenile fish, comprises feeding the growing fish live prey organismsthat have been fed a diet comprising an effective amount of purifiedpreen oil to increase a preen oil lipid content of the live preyorganisms. In one aspect, the growing fish are in aquaculture. Amountsof purified preen oil in the diet of the live prey organisms include 0.1wt to 50 wt %, specifically 1 wt % to 10 wt %, purified preen oil basedon the total weight of the diet. Live prey organisms include Anemia,rotifers and zooplankton. The diet of live prey organisms can containsimilar components to the basal fish feed compositions described herein.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1: Purification of Preen Oil from Preen Glands

Preen glands were removed from tails of laying hens and broilers usingmechanical means, cut open, and gland contents were removed. The preenlipids were extracted from the gland contents by heating to an internaltemperature of 80° C. and centrifugation at 3,000×g for 40 minutes, andthe fractions were analyzed using thin layer chromatography and gaschromatography (GC) for fatty acid composition. The amount of preen oilisolated from laying hens varied from 0.09-0.79 g of oil per preengland. The amount of preen oil isolated from broilers varied from0.05-0.21 g of oil per preen gland. The fatty acid content of purifiedpreen oil is provided in FIG. 1.

Preen oil was also isolated from turkey preen glands and the amount ofpreen oil is given in table 1.

TABLE 1 Preen oil isolated from turkey preen glands Mean Weight Numberof Samples of Total Oil Type of Sample Collected Material Per GlandYoung Turkey raised at UW 13 0.13 Madison Turkey Toms 10 0.73

Example 2: Anti-Inflammatory Activity of Purified Preen Oil in CollagenInduced Arthritis Mouse Model Methods:

Collagen-induced arthritis model. Four-week-old male DBA/1 mice (n=72)were purchased (Harlan, Indianapolis, Ind.) and housed in shoebox units(3 mice per box) with a 12:12 hour light-dark cycle. Mice were fed astandard rodent diet (8604, Harlan, 24.5% protein, 4.4% fat, 46.6%nitrogen-free extract) during a 7 day environmental acclimation period.After the acclimation period, mice were switched to a 97% complete,casein-based, semi-purified diet (based on the AIN-76A diet; TD140724)supplemented with 3% corn oil (CO) as the principle dietary fat (100%nutritionally replete after 3% CO addition). CO has been used as areference dietary lipid source in multiple collagen-induced arthritisstudies. In some studies, canola oil (CA) was used in place of corn oil.

After 3 weeks of maintenance on the CO diet, mice were immunized aspreviously described to induce arthritis. Briefly, 8 week old mice wererandomized into two groups for immunization against chick type IIcollagen (tIIc, Chondrex, Redmond, Wash., n=45), or 0.05M acetic acid(sham non-arthritic, n=27) emulsified with complete Freund's adjuvant (4mg/ml M tuberculosis H37RA). After a booster injection 3 weeks later,mice were monitored daily for clinical signs of arthritis (see clinicalarthritic score). In order to prevent excessive handling, arthritic micewere assessed for severity three times per week after diagnosis.

When the first mouse in a cage developed positive clinical signs ofarthritis (designated day 0), it was randomly assigned to one of thefollowing dietary treatments: (1) 3% CO diet, (2) 3% CA diet, or (3) 3%preen oil diet. Because the onset of arthritis occurs unpredictably,dietary treatments were initiated post-onset only. Specifically, thefirst arthritic mouse in a box was randomly reassigned to a dietarytreatment in a new cage. The remaining mice in the original cage wererelocated to the new cage only after development of arthritis. Arthriticand sham-immunized mice (maintained on the CO diet) remained on dietarytreatments for 63 days. Throughout the dietary treatment period,clinical arthritic score (described below) was monitored three times perweek, per mouse. Additionally, paw thickness measurements (describedbelow) were recorded once per week upon and post-development ofarthritis.

Clinical Arthritic Score.

Mice were examined three times per week by a trained observer blinded totreatment as detailed previously. Individual paws were assigned a scorebased on the following criteria: 0=normal: no inflammation; 1=mild:definite redness and swelling of the ankle or wrist or apparent rednessand swelling limited to individual digits, regardless of the number ofaffected digits; 2=moderate: redness and swelling of ankle and wrist;3=severe redness and swelling of the entire paw including digits;4=maximally inflamed limb involving multiple joints. The sum of all fourpaws was calculated, averaged between the two observers, and reported asthe “clinical arthritic score”.

Paw Thickness Measurements.

Immediately upon onset of arthritis and weekly thereafter, individualpaw thickness was recorded using a pressure sensitive caliper (SPI,Garden Grove, Calif.) as an adjunct to clinical arthritic score.

Paw Tissue Preparation.

On day 63 immediately after mice were euthanized by exsanguination,front and hind paws were harvested, snap frozen in liquid nitrogen, andseparately weighed then crushed under liquid nitrogen. Total protein wasextracted by tissue homogenization using T-PER® tissue proteinextraction reagent with Halt™ protease inhibitor cocktail (Thermo,Rockford, Ill.) including the following proteases: AEBSF (1 mM),Aprotinin (800 nM), Bestatin (50 uM), E64 (15 uM), Leupeptin (20 uM),Pepstatin A (10 uM), and EDTA (5 mM) at 10:1 v/w buffer to paw tissue.Samples were incubated on ice for 1 hour and centrifuged at 3,000 RPMfor 10 minutes at 4° C. Supernatant was collected and centrifuged at16,000×g at 4° C. for 45 minutes. Avoiding the top lipid layer,supernatant was collected and stored at −80° C. Total protein contentwas determined using a BCA protein assay (Thermo, Rockford, Ill.) priorto cytokine quantitation. Samples were diluted to a final proteinconcentration less than 1 mg/ml.

Cytokine analysis. Paw tissue concentrations of interleukin (IL)-1β,IL-6, IL-10 and tumor necrosis factor (TNF)-α were measured using themouse Bio-Plex Pro™ cytokine assay system according to themanufacturer's specifications (Bio-Rad Laboratories, Hercules, Calif.).Fluorescence was measured using the Luminex 100™ system (Bio-RadLaboratories) and results were analyzed using Bio-Plex Manager™ software(Bio-Rad Laboratories). Cytokine concentrations for each tissue werenormalized to the respective total protein content for that sample andreported as ng/g of tissue protein.

Fatty Acid Determination.

Total lipids from both dietary fat sources and paws were extracted usingchloroform/methanol (2:1 v/v) according to methods known in the art,with the exception that paws were crushed and cut into approximately 3mm×3 mm pieces prior to homogenization. Fatty acid methyl esters wereprepared by acid-catalyzed methylation. Relative percentages of fattyacid methyl esters were determined using an Agilent 6890N GC (AgilentTechnologies, Santa Clara, Calif.) equipped with a Restek Rt®-2560 100mbiscyanopropyl polysiloxane column (Restek, Bellefonte, Pa.).

Results:

As shown in FIG. 2, dietary preen oil supplemented at 3% (w/w) reducedthe clinical arthritic score in the collagen induced murine model ofrheumatoid arthritis. DBA/1 mice were fed diets containing 6% total fatsupplemented with either 3% corn oil or 3% preen oil for 63 dayspost-onset of arthritis. A blinded observer scored severity of arthritisthree times per week. Each point represents group means at each day; 3%preen oil (n=7), 3% corn oil (n=5). FIG. 3 shows the average reductionin arthritic severity over a 63-day period post onset of disease.Dietary preen oil fed at 3% w/w reduced arthritic severity 55% comparedto a 3% corn oil supplemented diet. Values are means±standard deviation.

FIG. 4 shows a comparison of 3% preen oil and corn oil diet fatty acidcompositions. Diets contained 6% total fat with corn oil making up thebasal fat. Means are grams of fatty acid per 100 g total fat±SEM. Notpictured: 3% preen oil diet contains a total of 8% of saturated fattyacids 5:0-12:0. Table 2 shows the hind paw fatty acid composition ofsham mice fed 3% preen oil or 3% corn oil over a 63 day period. Table 2shows that after oral administration of preen oil FAs not present incorn oil, such as 12:0, 17:0, 17:1, and 19:0, are increased in the pawsof mice suggesting that orally administered preen oil accumulates at thesite of inflammation.

TABLE 2 Hind paw fatty acid composition of sham mice fed 3% preen oil or3% corn oil over a 63 day period. 3% Corn Oil 3% Preen Oil Fatty acidg/100 g FA g/100 g FA Pooled SEM 12:0 ND 0.04 0.01 14:0 0.73 1.50 0.0414:1 0.10 0.22 0.01 15:0 0.01 0.07 0.01 16:0 8.92 11.71 0.47 16:1 13.9218.30 0.72 17:0 ND 0.09 0.01 17:1 ND 0.27 0.01 18:0 0.58 0.76 0.04 18:1c9 40.83 39.40 0.38 18:1 c11 3.04 3.43 0.08 18:2 n-6 29.64 21.37 0.4718:2 c9t11-CLA 0.06 ND 0.01 18:3n-3 0.19 0.18 0.02 19:0 ND 0.05 0.0120:0 0.10 0.12 0.01 20:1n-9 0.37 0.39 0.01 20:2n-6 0.04 0.05 0.0120:4n-6 0.28 0.33 0.01 22:6n-3 0.07 0.07 0.01 Unknown 1.42 1.70 0.22Total Identified 98.58 98.30 0.03 Values are means (n = 3/group). ND,non detectable

After 9 weeks of arthritis monitoring, paws were examined forproinflammatory cytokine levels. FIG. 5 shows that 3% preen oil reducedIL-1β to much lower levels than canola oil, and close to the levels seenin non-arthritic mice. FIG. 6 shows that 3% preen oil reduced IL-6 tomuch lower levels than canola oil, and lower than the levels seen innon-arthritic mice. These data are consistent with reduced arthritis inmice fed preen oil in that paw proinflammatory cytokines were reduced tosham levels in these mice.

Example 5: Effects of Preen Oil on Mouse Growth

8 week old, weight-matched mice were fed either a 3% CA diet or a 3%preen oil diet and weighed weekly over a 63-day period. As shown in FIG.7, mice fed 3% preen oil had a 30% increase in weight gain vs mice fed3% CA, suggesting that dietary preen oil increases growth rate or weightgain. Upon further examination several weeks later, increased growthrate did not result in an increased fat mass compared to mice fed 3% CAdiet. This observation suggests preen oil may increase growth ratewithout increased fat deposition in adipose or an associatedlipodystrophy.

Example 4: Effects of Preen Oil on the Growth and Survival of LarvalFathead Minnows Fed Exclusively on Formulated Diet Methods:

Experimental System.

Eight 7-L glass aquariums (tank water volume was 6 L) with flow-throughwater (carbon-filtered City of Madison, Wis. water). The temperature was25±0.5° C. The flow rate into each tank was 250 ml/min (2.5 tankturnovers per hour). Each tank had an air stone.

Fish.

Fifty newly hatched fathead minnows were added to each tank on day 0.The fish were obtained from the colony maintained at the UW-MadisonAquaculture Research Laboratory.

Feed.

Larval fathead minnows are typically fed live artemia, but for thisexperiment the fish were only fed dry, formulated diet (Skretting, GemmaMicro 150) to ensure that all fish fed on the treated diet.

Experimental Design.

A randomized block design was used for the experiment. There were twoblocks (upper or lower shelf), and two treatment groups: (1) control and(2) fed with diet top-coated with 2% preen oil. There were four tanksper treatment group and treatments were randomized within block. Thefish in each tank were fed twice daily (morning and afternoon) an excessof feed. Tanks were cleaned approximately weekly or as needed.

Data Collection.

The lengths of the fish were measured on day 0, 1 and 2 months usingdigital photos taken of each tank and a digital ruler (Pixelstick).

Results:

At month one, the larval fish fed the control and 2% preen oil dietswere 0.72±0.6 and 0.92±0.04 cm in length, respectively (FIG. 8). Thisdifference was significant at P=0.03 (paired, two-tailed t-test). Atmonth two, the larval fish fed the control and 2% preen oil diets were1.51±0.02 and 1.84±0.02 cm in length, respectively. This difference wassignificant at P=0.05 (paired, two-tailed t-test). Larval survival atthe end of the experiment in the control and 2% preen oil groups were16.5±3.1% and 41.5±9.1%, respectively (FIG. 9). Thus, the diet includingpreen oil increased larval fish growth measured as total length and alsosignificantly improved survival after one and two months.

Example 5: Effects of Preen Oil on the Survival of Hypoxic JuvenileWalleye Methods:

Experimental System.

Fifteen 7-L glass aquariums (tank water volume was 6 L) withflow-through water (carbon-filtered City of Madison, Wis. water). Thetemperature was 25±0.5° C. The flow rate into each tank was 250 ml/min(2.5 tank turnovers per hour).

Experimental Design.

A randomized design was used. There were three treatment groups: (1)control, (2) tallow oil iso-caloric control, and (3) 2% preen oil. Therewere five tanks per treatment group, each stock with five juvenilewalleye (approximately 2.3 g each at the start of experiment). The fishin each tank were fed twice daily (morning and afternoon) at a rate ofapproximately 5% of their body weight daily. The fish were fed(Skretting, Gemma Diet, 1 mm).

Results

Fifteen days after the start of the experiment, the water to the fishtanks was turned off to prevent a change in water temperature duringrepairs to the lab's hot water system. The air to the tanks, however,was also mistakenly turned off and the environmental conditions in thetanks rapidly became hypoxic. By the time this was discovered, many fishhad died or were severely stressed. The percent survival by treatmentgroup was as follows: Control (40%), Iso-caloric tallow oil control(52%), preen oil (96%) (FIG. 10). The differences were highlysignificant (P<0.02 between preen oil and control and preen oil andiso-caloric control). The average fish weight of the fish on day 15 was3.6+0.1 g. There was no significant treatment effect on growth at thistime.

The use of the terms “a” and “an” and “the” and similar referents(especially in the context of the following claims) are to be construedto cover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms first, second etc.as used herein are not meant to denote any particular ordering, butsimply for convenience to denote a plurality of, for example, layers.The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted. Recitation of ranges of values aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range, unless otherwiseindicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. The endpointsof all ranges are included within the range and independentlycombinable. All methods described herein can be performed in a suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”), is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention as used herein.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims. Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

1-25. (canceled)
 26. A method of feeding a growing fish, comprisingfeeding the growing fish a fish food composition comprising an effectiveamount of purified preen oil to improve survival and/or stimulate growthin the growing fish.
 27. The method of claim 26, wherein the foodcomposition comprises 0.01 to 10 wt % purified preen oil on a w/w basis.28. The method of claim 26, wherein the fish is in aquaculture.
 29. Themethod of claim 26, wherein the fish is a finfish or a shellfish. 30.The method of claim 29, wherein the fish is carp, tilapia, hybridstriped bass, salmon, trout, catfish, yellow perch, walleye, cod, cobia,sea bass, tuna, sole, shrimp, scallops or oysters.
 31. The method ofclaim 26, wherein growth in the growing fish is the average length of apopulation of growing fish.
 32. The method of claim 32, wherein thegrowing fish is a larval or juvenile fish.
 33. The method of claim 32,wherein the food composition comprises 0.01 to 10 wt % purified preenoil on a w/w basis.
 34. The method of claim 32, wherein the fish is inaquaculture.
 35. The method of claim 32, wherein the fish is a finfishor a shellfish.
 36. The method of claim 35, wherein the fish is carp,tilapia, hybrid striped bass, salmon, trout, catfish, yellow perch,walleye, cod, cobia, sea bass, tuna, sole, shrimp, scallops or oysters.37. The method of claim 32, wherein growth in the growing fish is theaverage length of a population of growing fish.
 38. A method of reducinga risk of hypoxia in a fish in aquaculture, comprising feeding the fisha fish food composition comprising an effective amount of purified preenoil to reduce the risk of hypoxia in the fish in aquaculture.
 39. Themethod of claim 38, wherein the food composition comprises 0.01 to 10 wt% purified preen oil on a w/w basis.
 40. The method of claim 38, whereinthe fish is a finfish or a shellfish.
 41. The method of claim 40,wherein the fish is carp, tilapia, hybrid striped bass, salmon, trout,catfish, yellow perch, walleye, cod, cobia, sea bass, tuna, sole,shrimp, scallops or oysters.
 42. The method of claim 38, wherein thefish is a larval or juvenile fish.
 43. The method of claim 38, whereinthe food composition comprises 0.01 to 10 wt % purified preen oil on aw/w basis.
 44. A method of feeding live prey organisms for use as foodin aquaculture, comprising feeding the live prey organisms a dietcomprising an effective amount of purified preen oil to increase a preenoil lipid content of the live prey organisms.
 45. The method of claim44, wherein the diet comprises 0.1 wt % to 50 wt % of purified preen oilbased on the total weight of the diet.
 46. A method of feeding a growingfish, comprising feeding the growing fish live prey organisms that havebeen fed a diet comprising an effective amount of purified preen oil toincrease a preen oil lipid content of the live prey organisms.
 47. Themethod of claim 46, wherein the growing fish are larval or juvenilefish.
 48. The method of claim 46, wherein the diet of the prey organismcomprises 0.1 wt % to 50 wt % of purified preen oil based on the totalweight of the diet.
 49. The method of claim 48, wherein the growing fishare in aquaculture.
 50. The method of claim 48, wherein the live preyorganisms are Artemia, rotifers or zooplankton.
 51. (canceled)